1. Field of the Invention
The present invention relates to a method of producing urokinase preparations. In more particular, the invention is concerned with a method of preventing the degradation of urokinase to be caused when an aqueous solution of urokinase is for example heated at 60.degree. C. for 10 hours to inactivate pathogenic viruses.
2. Description of the Related Art
Urokinase is a plasminogen activating factor which exists in small quantities in urine and, with its fibrinolytic activity, has nowadays been put in wide use for the therapy of thrombosis. Urokinase dates as far back as 1800's when the existence in urine of a substance having fibrinolytic activity was already recognized by Stahli, Gehrig, et al. As recently as 1952, the plasminogen activator was designated "urokinase" (hereinafter referred to briefly as "UK") by Sobel. In 1957, Ploug nearly completed the method of determining activity of this substance as well as the procedure of separating and purifying the same from urine, thereby laying down the foundation for its clinical application to the therapy of thrombosis [Ploug, J.: Biochim. Biophys. Acta, 24, 278 (1957)]. Thereafter, White clarified that UK is a substance consisting of two components but not a single component substance: namely, there exist high-molecular type urokinase (hereinafter referred to as "H-UK") having a molecular weight of 54,000 and low-molecular type urokinase (hereinafter referred to as "L-UK") having a molecular weight of 32,000, and it was found that the only the former is contained in urine immediately discharged from human body, whereas the latter is produced from the former through decomposition and cleavage by proteolytic enzymes such as uropepsin taking place during storage, extraction or purification of urine [White, W. F. et al: Biochemistry,, 5, 2160 (1966)]. Consequently, it can be said that the currently available urokinase preparations contain the mixtures of H-UK and L-UK, though their ratio varies widely.
Now referring to the above-described two kinds of urokinase, H-UK and L-UK, in terms of comparison of their performances, determination by way of the ordinary determination methods, such as the fibrin plate method, fibrin tube method [Ploug, J., et al.: Biochim. Biophys. Acta, 24, 278 (1957)] or AGLME synthetic substrate method [Johnson, A. J. et al.: Thromb. Diath. Haemorrh., 21, 259 (1969)], indicates that the pure compounds of H-UK and L-HK show approximately the same level of activity per mmole, but on the other hand, Chandler-Poole method [Chandler, A. B.: Lab. Invest., 7, 110 (1957). Poole, J. C. F.: Q. J. Exp. Physiol., 44, 372 (1959)] which is the nearest to the thrombolytic action in vivo and the method using the naturally occurring compound of glutamylplasminogen (Japanese Patent Application Laid-open No. 130485/1978) demonstrate that H-UK exhibits a level of activity nearly double that of L-UK. This means that H-UK, when put into clinical application in almost half an amount can produce the effects equal to L-UK, and can prevent unexpected side-effects such as bleeding (Japanese Patent Application Laid-open No. 130485/1978). H-UK is known to provide a variety of additional advantages, and currently, in Europe and America as well as in Japan, the standards covering the urokinase preparations specify that the content of H-UK is not to be less than 75%.
On the other hand, it is a matter of common knowledge that hepatitis virus and other pathogenic viruses are present in human blood, organs and urine. Therefore, the UK preparations produced from human urine, when administered to human being as such, provides the possibility of being affected with viral infections. Both in U.S.A. and Japan at present, the obligation has been placed to perform heat treatment at 60.degree. C. for 10 hours of the UK preparations for the purpose of inactivation of pathogenic viruses.
Since such heat treatment at 60.degree. C. for 10 hours of UK solutions results in reduction of activity of UK, however, various means have been conducted into practice for the prevention of reduction of activity. Actually adopted are the methods of adding on the occasion of heat treatment amino acids, saccharides, salts, gelatin, human serum albumin, hydroxypropylcellulose, etc. The Japanese Patent Application Laid-Open Nos. 43233/1981, 142592/1978 and 2391/1981). The above-described methods can prevent reduction of the UK activity to be caused by heat treatment at 60.degree. C. for 10 hours but only to a limited degree.